Single-Cell Transcriptomics and Fate Mapping of Ependymal Cells Reveals an Absence of Neural Stem Cell Function.

Ependymal cells are multi-ciliated cells that form the brain's ventricular epithelium and a niche for neural stem cells (NSCs) in the ventricular-subventricular zone (V-SVZ). In addition, ependymal cells are suggested to be latent NSCs with a capacity to acquire neurogenic function. This remains highly controversial due to a lack of prospective in vivo labeling techniques that can effectively distinguish ependymal cells from neighboring V-SVZ NSCs. We describe a transgenic system that allows for targeted labeling of ependymal cells within the V-SVZ. Single-cell RNA-seq revealed that ependymal cells are enriched for cilia-related genes and share several stem-cell-associated genes with neural stem or progenitors. Under in vivo and in vitro neural-stem- or progenitor-stimulating environments, ependymal cells failed to demonstrate any suggestion of latent neural-stem-cell function. These findings suggest remarkable stability of ependymal cell function and provide fundamental insights into the molecular signature of the V-SVZ niche.

Prx2 (Peroxiredoxin 2) as a Cause of Hydrocephalus After Intraventricular Hemorrhage.

Background and Purpose- Our recent study demonstrated that release of Prx2 (peroxiredoxin 2) from red blood cells (RBCs) is involved in the inflammatory response and brain injury after intracerebral hemorrhage. The current study investigated the role of extracellular Prx2 in hydrocephalus development after experimental intraventricular hemorrhage. Methods- There were 4 parts in this study. First, Sprague-Dawley rats received an intraventricular injection of lysed RBC or saline and were euthanized at 1 hour for Prx2 measurements. Second, rats received an intraventricular injection of Prx2, deactivated Prx2, or saline. Third, lysed RBC was coinjected with conoidin A, a Prx2 inhibitor, or vehicle. Fourth, rats received Prx2 injection and were treated with minocycline or saline (i.p.). The effects of Prx2 and the inhibitors were examined using magnetic resonance imaging assessing ventriculomegaly, histology assessing ventricular wall damage, and immunohistochemistry to assess inflammation, particularly at the choroid plexus. Results- Intraventricular injection of lysed RBC resulted in increased brain Prx2 and hydrocephalus. Intraventricular injection of Prx2 alone caused hydrocephalus, ventricular wall damage, activation of choroid plexus epiplexus cells (macrophages), and an accumulation of neutrophils. Conoidin A attenuated lysed RBC-induced injury. Systemic minocycline treatment reduced the epiplexus cell activation and hydrocephalus induced by Prx2. Conclusions- Prx2 contributed to the intraventricular hemorrhage-induced hydrocephalus, probably by inducing inflammatory responses in choroid plexus and ventricular wall damage.

Morphological analysis for neuronal pathway from the hindbrain ependymocytes to the hypothalamic kisspeptin neurons.

Hindbrain ependymocytes are postulated to have a glucose-sensing role in regulating gonadal functions. Previous studies have suggested that malnutrition-induced suppression of gonadotropin secretion is mediated by noradrenergic inputs from the A2 region in the solitary tract nucleus to the paraventricular nucleus (PVN), and by corticotropin-releasing hormone (CRH) release in the hypothalamus. However, no morphological evidence to indicate the neural pathway from the hindbrain ependymocytes to hypothalamic kisspeptin neurons, a center for reproductive function in mammals, currently exists. The present study aimed to examine the existence of a neuronal pathway from the hindbrain ependymocytes to kisspeptin neurons in the arcuate nucleus (ARC) and anteroventral periventricular nucleus (AVPV). To determine this, wheat-germ agglutinin (WGA), a trans-synaptic tracer, was injected into the fourth ventricle (4V) in heterozygous Kiss1-tandem dimer Tomato (tdTomato) rats, where kisspeptin neurons were visualized by tdTomato fluorescence. 48 h after the WGA injection, brain sections were taken from the forebrain, midbrain and hindbrain and subjected to double immunohistochemistry for WGA and dopamine ß-hydroxylase (DBH) or CRH. WGA immunoreactivities were found in vimentin-immunopositive ependymocytes of the 4V and the central canal (CC), but not in the third ventricle. The WGA immunoreactivities were detected in some tdTomato-expressing cells in the ARC and AVPV, DBH-immunopositive cells in the A1-A7 noradrenergic nuclei, and CRH-immunopositive cells in the PVN. These results suggest that the hindbrain ependymocytes have neuronal connections with the kisspeptin neurons, most probably via hindbrain noradrenergic and CRH neurons to relay low energetic signals for regulation of reproduction.

Chronic Toxicology Studies of Basal Insulin Peglispro in Rats and Dogs: A Novel, PEGylated Insulin Lispro Analog with a Prolonged Duration of Action.

Basal insulin peglispro (BIL) consists of insulin lispro with a 20-kDa polyethylene glycol (PEG) moiety covalently attached to lysine B28. Because chronic parenteral administration of PEGylated proteins to animals has sometimes resulted in PEG vacuolation of tissue macrophages, renal tubular cells, and choroid plexus ependymal cells, we investigated whether chronic subcutaneous (sc) injection of BIL in rats (52 weeks) and dogs (39 weeks) was associated with systemic toxicities or other changes, including vacuolation of tissue macrophages, renal tubular cells, and ependymal cells. Rats and dogs received daily sc injections of BIL (rats: 0.17, 0.45, or 1.15 mg/kg/d and dogs: 0.025, 0.10, or 0.20 mg/kg/d) and the reference compound, HUMULIN N® (neutral protamine Hagedorn [NPH] human insulin; rats: 0.15 mg/kg/d and dogs: 0.02-0.03 mg/kg/d). Animals were evaluated for standard end points including mortality, clinical signs, body weights, toxicokinetics, glucodynamics, clinical pathology, and morphological pathology. Nonadverse injection site lipohypertrophy occurred for all BIL and NPH doses but more frequently with BIL. No BIL-related hyperplasia or neoplasia was observed. There was no vacuolation of tissue macrophages, renal tubular cells, or ependymal cells attributable to PEG. These studies demonstrate BIL is not associated with tissue vacuolation attributable to PEG at 4- to 6-fold multiple of the median clinical exposure in patients with diabetes.

Acetazolamide Attenuates Thrombin-Induced Hydrocephalus.

Our previous studies demonstrated that thrombin is an important factor in brain injury after intracerebral and intraventricular hemorrhage. This study examined the effect of acetazolamide, a carbonic anhydrase inhibitor, on thrombin-induced hydrocephalus. There were two parts in this study. First, rats had an injection of either 50 µl saline or 3 U thrombin into the right lateral ventricle. Second, rats had an injection of 3 U thrombin into the right lateral ventricle and were treated with either vehicle or acetazolamide (30 mg/kg, intraperitoneally (IP)) at 1 h after thrombin infusion. Lateral ventricle volumes were measured in magnetic resonance imaging T2 images and the brains were used for histology analysis at 24 h later. Intraventricular injection of thrombin induced significantly larger ventricle volume (27.8 ± 3.7 vs 8.5 ± 1.3 mm(3), n = 6, p < 0.01) and more ventricular wall damage (the breakdown of the ependymal layer, 20.2 ± 3.1 vs 2.4 ± 0.8 %, n = 6, p < 0.01) compared with saline injection. Acetazolamide treatment (30 mg/kg, IP) markedly attenuated thrombin-induced hydrocephalus (16.1 ± 4.2 mm(3) vs 29.5 ± 5.3 mm(3), n = 6, p < 0.01). These results suggest decreasing CSF production by acetazolamide attenuated thrombin-induced hydrocephalus in rats.

Decorin prevents the development of juvenile communicating hydrocephalus.

In post-haemorrhagic and other forms of communicating hydrocephalus, cerebrospinal fluid flow and drainage is obstructed by subarachnoid fibrosis in which the potent fibrogenic cytokine transforming growth factor-ß has been aetiologically implicated. Here, the hypothesis that the transforming growth factor-ß antagonist decorin has therapeutic potential for reducing fibrosis and ventriculomegaly was tested using a rat model of juvenile communicating hydrocephalus. Hydrocephalus was induced by a single basal cistern injection of kaolin in 3-week-old rats, immediately followed by 3 or 14 days of continuous intraventricular infusion of either human recombinant decorin or phosphate-buffered saline (vehicle). Ventricular expansion was measured by magnetic resonance imaging at Day 14. Fibrosis, transforming growth factor-ß/Smad2/3 activation and hydrocephalic brain pathology were evaluated at Day 14 and the inflammatory response at Days 3 and 14 by immunohistochemistry and basic histology. Analysis of ventricular size demonstrated the development of hydrocephalus in kaolin-injected rats but also revealed that continuous decorin infusion prevented ventricular enlargement, such that ventricle size remained similar to that in intact control rats. Decorin prevented the increase in transforming growth factor-ß1 and phosphorylated Smad2/3 levels throughout the ventricular system after kaolin injection and also inhibited the deposition of the extracellular matrix molecules, laminin and fibronectin in the subarachnoid space. In addition, decorin protected against hydrocephalic brain damage inferred from attenuation of glial and inflammatory reactions. Thus, we conclude that decorin prevented the development of hydrocephalus in juvenile rats by blocking transforming growth factor-ß-induced subarachnoid fibrosis and protected against hydrocephalic brain damage. The results suggest that decorin is a potential clinical therapeutic for the treatment of juvenile post-haemorrhagic communicating hydrocephalus.

Infant satiety depends on transient expression of cholecystokinin-1 receptors on ependymal cells lining the third ventricle in mice.

Cholecystokinin (CCK) is a hypothetical controller for suckling and infancy body weight, although the underlying mechanisms remain unclear. Therefore, the present study analysed the mechanisms using mice lacking the CCK-1 receptor (CCK1R-/-). Although CCK1R-/- mice displayed normal weights at birth and adulthood, CCK1R-/- pups had enlarged adipocytes and were overweight from the first to second week after birth, regardless of maternal genotype. The lacZ reporter gene assay and/or calcium imaging analysis demonstrated that CCK-1 receptors were abundant in satiety-controlling regions such as the hypothalamus, brainstem, nodose ganglion and pylorus in adults, whereas these signals were few to lacking at pre-weanling stages. At postnatal day (PD) 6, the increase in cFos expression in the medullary nucleus tractus solitarius was similarly triggered by gastrointestinal milk- or saline filling in both genotypes, further indicating immature CCK-1 receptor function in an ascending satiety-controlling system during infancy. Conversely, third ventricle ependymal tanycyte-like cells expressed CCK-1 receptors with expression peaking at PD6. At PD6, wild-type but not CCK1R-/- mice had increased cFos immunoreactivity in ependymal cells following gastrointestinal milk filling whereas the response became negligible at PD12. In addition, ependymal cFos was not increased by saline filling, indicating that these responses are dependent on CCK-1 receptors, developmental stage and nutrients. Furthermore, body weights of wild-type pups were transiently increased by blocking ependymal CCK receptor function with microinjection of a CCK-1 antagonist, but not a CCK-2 antagonist. Hence, we demonstrate de novo functions of ependymal CCK-1 receptors and reveal a new aspect of infant satiety-controlling mechanisms.

γ-Aminobutyric acid-ρ expression in ependymal glial cells of the mouse cerebellum.

The ependymal glial cells (EGCs) from the periventricular zone of the cerebellum were studied to determine their distribution and the functional properties of their γ-aminobutyric acid type A (GABA(A) ) receptors. EGCs were identified by the presence of ciliated structures on their ventricular surface and their expression of glial fibrillary acidic protein (GFAP). Interestingly, diverse cell types, including neurons, astrocytes, and other types of glia, were identified in the subventricular zone by their current profiles. Electron microscopy showed ciliated cells and myelinated axons in this zone, but we found no collateral connections to suggest the presence of functional synapses. GABA-mediated currents were recorded from EGCs in cerebellar slices from postnatal days 13 to 35 (PN13-PN35). These currents were blocked by TPMPA (a highly specific GABA(A) ρ subunit antagonist) and bicuculline (a selective antagonist for classic GABA(A) receptors). Pentobarbital failed to modulate GABA(A)-mediated currents despite the expression of GABAα1 and GABAγ2 subunits. In situ hybridization, RT-PCR, and immunofluorescence studies confirmed GABAρ1 expression in EGCs of the cerebellum. We conclude that cerebellar EGCs express GABAρ1, which is functionally involved in GABA(A) receptor-mediated responses that are unique among glial cells of the brain.

FM19G11 favors spinal cord injury regeneration and stem cell self-renewal by mitochondrial uncoupling and glucose metabolism induction.

Spinal cord injury is a major cause of paralysis with no currently effective therapies. Induction of self-renewal and proliferation of endogenous regenerative machinery with noninvasive and nontoxic therapies could constitute a real hope and an alternative to cell transplantation for spinal cord injury patients. We previously showed that FM19G11 promotes differentiation of adult spinal cord-derived ependymal stem cells under hypoxia. Interestingly, FM19G11 induces self-renewal of these ependymal stem cells grown under normoxia. The analysis of the mechanism of action revealed an early increment of mitochondrial uncoupling protein 1 and 2 with an early drop of ATP, followed by a subsequent compensatory recovery with activated mitochondrial metabolism and the induction of glucose uptake by upregulation of the glucose transporter GLUT-4. Here we show that phosphorylation of AKT and AMP-activated kinase (AMPK) is involved in FM19G11-dependent activation of GLUT-4, glucose influx, and consequently in stem cell self-renewal. Small interfering RNA of uncoupling protein 1/2, GLUT-4 and pharmacological inhibitors of AKT, mTOR and AMPK signaling blocked the FM19G11-dependent induction of the self-renewal-related markers Sox2, Oct4, and Notch1. Importantly, FM19G11-treated animals showed accelerated locomotor recovery. In vivo intrathecal sustained administration of FM19G11 in rats after spinal cord injury showed more neurofilament TUJ1-positive fibers crossing the injured area surrounded by an increase of neural precursor Vimentin-positive cells. Overall, FM19G11 exerts an important influence on the self-renewal of ependymal stem progenitor cells with a plausible neuroprotective role, providing functional benefits for spinal cord injury treatment.

Targeting choroid plexus epithelia and ventricular ependyma for drug delivery to the central nervous system.

BACKGROUND: Because the choroid plexus (CP) is uniquely suited to control the composition of cerebrospinal fluid (CSF), there may be therapeutic benefits to increasing the levels of biologically active proteins in CSF to modulate central nervous system (CNS) functions. To this end, we sought to identify peptides capable of ligand-mediated targeting to CP epithelial cells reasoning that they could be exploited to deliver drugs, biotherapeutics and genes to the CNS. METHODS: A peptide library displayed on M13 bacteriophage was screened for ligands capable of internalizing into CP epithelial cells by incubating phage with CP explants for 2 hours at 37C and recovering particles with targeting capacity. RESULTS: Three peptides, identified after four rounds of screening, were analyzed for specific and dose dependent binding and internalization. Binding was deemed specific because internalization was prevented by co-incubation with cognate synthetic peptides. Furthermore, after i.c.v. injection into rat brains, each peptide was found to target phage to epithelial cells in CP and to ependyma lining the ventricles. CONCLUSION: These data demonstrate that ligand-mediated targeting can be used as a strategy for drug delivery to the central nervous system and opens the possibility of using the choroid plexus as a portal of entry into the brain.

Postnatal exposure to N-ethyl-N-nitrosurea disrupts the subventricular zone in adult rodents.

N-ethyl-N-nitrosurea (ENU), a type of N-nitrous compound (NOC), has been used as inductor for brain tumours due to its mutagenic effect on the rodent embryo. ENU also affected adult neurogenesis when administered during pregnancy. However, no studies have investigated the effect of ENU when exposured during adulthood. For this purpose, three experimental groups of adult mice were injected with ENU at different doses and killed shortly after exposure. When administered in adult mice, ENU did not form brain tumours but led to a disruption of the subventricular zone (SVZ), an adult neurogenic region. Analyses of the samples revealed a reduction in the numbers of neural progenitors compared with control animals, and morphological changes in ependymal cells. A significant decrease in proliferation was tested in vivo with 5-bromo-2-deoxyuridine administration and confirmed in vitro with a neurosphere assay. Cell death, assessed as active-caspase-3 reactivity, was more prominent in treated animals and cell death-related populations increased in parallel. Two additional groups were maintained for 45 and 120 days after five doses of ENU to study the potential regeneration of the SVZ, but only partial recovery was detected. In conclusion, exposure to ENU alters the organization of the SVZ and causes partial exhaustion of the neurogenic niche. The functional repercussion of these changes remains unknown, but exposure to NOCs implies a potential risk that needs further evaluation.

Pigment epithelium-derived factor is a niche signal for neural stem cell renewal.

Adult stem cells are characterized by self-renewal and multilineage differentiation, and these properties seem to be regulated by signals from adjacent differentiated cell types and by extracellular matrix molecules, which collectively define the stem cell "niche." Self-renewal is essential for the lifelong persistence of stem cells, but its regulation is poorly understood. In the mammalian brain, neurogenesis persists in two germinal areas, the subventricular zone (SVZ) and the hippocampus, where continuous postnatal neuronal production seems to be supported by neural stem cells (NSCs). Here we show that pigment epithelium-derived factor (PEDF) is secreted by components of the murine SVZ and promotes self-renewal of adult NSCs in vitro. In addition, intraventricular PEDF infusion activated slowly dividing stem cells, whereas a blockade of endogenous PEDF decreased their cycling. These data demonstrate that PEDF is a niche-derived regulator of adult NSCs and provide evidence for a role for PEDF protein in NSC maintenance.

Subventricular zone-mediated ependyma repair in the adult mammalian brain.

The subventricular zone (SVZ) of the adult mouse brain is a narrow stem cell niche that lies along the length of the lateral wall of the lateral ventricles. The SVZ supports neurogenesis throughout adulthood; however, with increasing age, the ventral SVZ deteriorates and only the dorsolateral SVZ remains neurogenic. Associated with the elderly dorsolateral SVZ, we reported previously an increased number of astrocytes interposed within the adjacent ependymal lining. Here, we show that astrocytes integrated within the ependyma are dividing, BrdU-labeled astrocytes that share cellular adherens with neighboring ependymal cells. By tracking BrdU-labeled astrocytes over time, we observed that, as they incorporated within the ependyma, they took on antigenic and morphologic characteristics of ependymal cells, suggesting a novel form of SVZ-supported "regenerative" repair in the aging brain. A similar form of SVZ-mediated ependyma repair was also observed in young mice after mild ependymal cell denudation with low dosages of neuraminidase. Together, this work identifies a novel non-neuronal mechanism of regenerative repair by the adult SVZ.

Peripheral osmotic stimulation inhibits the brain's innate immune response to microdialysis of acidic perfusion fluid adjacent to supraoptic nucleus.

During the brain's innate immune response microglia, astroglia and ependymal cells resolve/repair damaged tissue and control infection. Released interleukin-1beta (IL-1beta) reaching cerebroventricles stimulates circumventricular organs (CVOs; subfornical organ, SFO; organum vasculosum lamina terminalis, OVLT), the median preoptic nucleus (MePO), and magnocellular and parvocellular neurons in the supraoptic (SON) and paraventricular (PVN) nuclei. Hypertonic saline (HS) also activates these osmosensory CVOs and neuroendocrine systems, but, in contrast to IL-1beta, inhibits the peripheral immune response. To examine whether the brain's innate immune response is attenuated by osmotic stimulation, sterile acidic perfusion fluid was microdialyzed (2 microl/min) in the SON area of conscious rats for 6 h with sterile HS (1.5 M NaCl) injected subcutaneously (15 ml/kg) at 5 h. Immunohistochemistry identified cytokine sources (IL-1beta(+); OX-42(+) microglia) and targets (IL-1R(+); inducible cyclooxygenase, COX-2(+); c-Fos(+)) near the probe, in CVOs, MePO, ependymal cells, periventricular hypothalamus, SON, and PVN. Inserting the probe stimulated magnocellular neurons (c-Fos(+); SON; PVN) via the MePO (c-Fos(+)), a response enhanced by HS. Microdialysis activated microglia (OX-42(+); amoeboid/hypertrophied; IL-1beta(+)) in the adjacent SON and bilaterally in perivascular areas of the PVN, periventricular hypothalamus and ependyma, coincident with c-Fos expression in ependymal cells and COX-2 in the vasculature. These microglial responses were attenuated by HS, coincident with activating parvocellular and magnocellular neuroendocrine systems and elevating circulating IL-1beta, oxytocin, and vasopressin. Acidosis-induced cellular injury from microdialysis activated the brain's innate immune response by a mechanism inhibited by peripheral osmotic stimulation.

Hydrogen peroxide at a concentration used during neurosurgery disrupts ciliary function and causes extensive damage to the ciliated ependyma of the brain.

OBJECTIVES: Hydrogen peroxide [H(2)O(2): 3% w/v (1.1 M)] has been used as a haemostatic agent during neurosurgery applied to both the external and ventricular surface of the brain. We hypothesised that H(2)O(2) would be toxic to the ciliated ependyma, a single layer of cells that separates cerebrospinal fluid from the neuronal tissue of the brain. MATERIALS AND METHODS: The effect of H(2)O(2) was assessed by determining ependymal ciliary beat frequency (CBF) using high-speed video analysis and ultrastructure by electron microscopy. RESULTS: Brief exposure to H(2)O(2) caused cessation of ciliary beat frequency and extensive damage of the ependyma. CONCLUSIONS: Damage to the ciliated ependyma is of concern, as regeneration following damage is very poor and if breached underlying neuronal tissue and a population of neuronal progenitor cells that lie immediately beneath may also be exposed to H(2)O(2).

Ependymal stem cells divide asymmetrically and transfer progeny into the subventricular zone when activated by injury.

Evidence is presented to show that cells of the ependymal layer surrounding the ventricles of the mammalian (rat) forebrain act as neural stem cells (NSCs), and that these cells can be activated to divide by a combination of injury and growth factor stimulation. Several markers of asymmetric cell division (ACD), a characteristic of true stem cells, are expressed asymmetrically in the ependymal layer but not in the underlying subventricular zone (SVZ), and when the brain is treated with a combination of local 6-hydroxydopamine (6-OHDA) with systemic delivery of transforming growth factor-alpha (TGFalpha), ependymal cells divide asymmetrically and transfer progeny into the SVZ. The SVZ cells then divide as transit amplifying cells (TACs) and their progeny enter a differentiation pathway. The stem cells in the ependymal layer may have been missed in many previous studies because they are usually quiescent and divide only in response to strong stimuli.

Methylprednisolone inhibits the proliferation of endogenous neural stem cells in nonhuman primates with spinal cord injury.

OBJECTIVE The aim of this work was to investigate the effects of methylprednisolone on the proliferation of endogenous neural stem cells (ENSCs) in nonhuman primates with spinal cord injury (SCI). METHODS A total of 14 healthy cynomolgus monkeys ( Macaca fascicularis) (4-5 years of age) were randomly divided into 3 groups: the control group (n = 6), SCI group (n = 6), and methylprednisolone therapy group (n = 2). Only laminectomy was performed in the control animals at T-10. SCI was induced in monkeys using Allen's weight-drop method (50 mm × 50 g) to injure the posterior portion of the spinal cord at T-10. In the methylprednisolone therapy group, monkeys were intravenously infused with methylprednisolone (30 mg/kg) immediately after SCI. All animals were intravenously infused with 5-bromo-2-deoxyuridine (BrdU) (50 mg/kg/day) for 3 days prior to study end point. The small intestine was dissected for immunohistochemical examination. After 3, 7, and 14 days, the spinal cord segments of the control and SCI groups were dissected to prepare frozen and paraffin sections. The proliferation of ENSCs was evaluated using BrdU and nestin immunofluorescence staining. RESULTS Histological examination showed that a larger number of mucosa epithelial cells in the small intestine of all groups were BrdU positive. Nestin-positive ependymal cells are increased around the central canal after SCI. After 3, 7, and 14 days of SCI, BrdU-positive ependymal cells in the SCI group were significantly increased compared with the control group, and the percentage of BrdU-positive cells in the left/right ventral horns and dorsal horn was significantly higher than that of the control group. Seven days after SCI, the percentages of both BrdU-positive ependymal cells around the central canal and BrdU- and nestin-double positive cells in the left/right ventral horns and dorsal horn were significantly lower in the methylprednisolone therapy group than in the SCI group. CONCLUSIONS While ENSCs proliferate significantly after SCI in nonhuman primates, methylprednisolone can inhibit the proliferation of ependymal cells after SCI.

Alcohol consumption impairs the ependymal cilia motility in the brain ventricles.

Ependymal cilia protrude into the central canal of the brain ventricles and spinal cord to circulate the cerebral spinal fluid (CSF). Ependymal cilia dysfunction can hinder the movement of CSF leading to an abnormal accumulation of CSF within the brain known as hydrocephalus. Although the etiology of hydrocephalus was studied before, the effects of ethanol ingestion on ependymal cilia function have not been investigated in vivo. Here, we report three distinct types of ependymal cilia, type-I, type-II and type-III classified based upon their beating frequency, their beating angle, and their distinct localization within the mouse brain-lateral ventricle. Our studies show for the first time that oral gavage of ethanol decreased the beating frequency of all three types of ependymal cilia in both the third and the lateral rat brain ventricles in vivo. Furthermore, we show for the first time that hydin, a hydrocephalus-inducing gene product whose mutation impairs ciliary motility, and polycystin-2, whose ablation is associated with hydrocephalus are colocalized to the ependymal cilia. Thus, our studies reinforce the presence of three types of ependymal cilia in the brain ventricles and demonstrate the involvement of ethanol as a risk factor for the impairment of ependymal cilia motility in the brain.

KCa3.1 channels modulate the processing of noxious chemical stimuli in mice.

Intermediate conductance calcium-activated potassium channels (KCa3.1) have been recently implicated in pain processing. However, the functional role and localization of KCa3.1 in the nociceptive system are largely unknown. We here characterized the behavior of mice lacking KCa3.1 (KCa3.1-/-) in various pain models and analyzed the expression pattern of KCa3.1 in dorsal root ganglia (DRG) and the spinal cord. KCa3.1-/- mice demonstrated normal behavioral responses in models of acute nociceptive, persistent inflammatory, and persistent neuropathic pain. However, their behavioral responses to noxious chemical stimuli such as formalin and capsaicin were increased. Accordingly, formalin-induced nociceptive behavior was increased in wild-type mice after administration of the KCa3.1 inhibitor TRAM-34. In situ hybridization experiments detected KCa3.1 in most DRG satellite glial cells, in a minority of DRG neurons, and in ependymal cells lining the central canal of the spinal cord. Together, our data point to a specific inhibitory role of KCa3.1 for the processing of noxious chemical stimuli.

ANP-mediated cGMP signaling and phosphodiesterase inhibition in the rat cervical spinal cord.

Natriuretic peptides (NP) and the corresponding receptors are present in the rodent spinal cord. We have studied the structures which respond to atrial natriuretic peptide, brain natriuretic peptide, or C-type natriuretic peptide with an increased synthesis of cGMP. NP-responsive cGMP-producing structures were observed in laminae I-III, and X, and in addition in ependymal cells, astrocytes and a subpopulation of dorsal root ganglion cells. As the cGMP concentration is controlled by the rate of synthesis and the rate of breakdown by phosphodiesterases, we studied NP-responsive structures in spinal cord slices incubated in the presence of different phosphodiesterase inhibitors. We studied EHNA and BAY 60-7550 as selective PDE2 inhibitors, sildenafil as a selective PDE5 inhibitors, dipyridamole as a mixed type PDE5 and PDE10 inhibitor, rolipram as a PDE4 inhibitor, and SCH 81566 as a selective PDE9 inhibitor. Double immunostainings showed that cGMP-IR colocalized partial with the vesicular acetylcholine transporter molecule in lamina X, with Substance P in a subpopulation of neuronal fibers situated dorsolateral, and with a subpopulation of CGRP-IR dorsal root ganglion neurons. Colocalization of cGMP-IR was absent with parvalbumin, synaptophysin, and the vesicular transporter molecules for GABA and glutamate. It is concluded that NPs in the spinal cord are probably involved in integrating intersegmental sensory processing in the spinal cord although the greater part of the NP-responsive cGMP-producing fibers could not be characterized. PDE2, 5, and 9 are involved in regulating NP-stimulated cGMP levels in the spinal cord. NPs may have a role in regulating cerebrospinal fluid homeostasis.